This invention relates to biocontrol of plant-pathogenic nematodes, and in particular to a monoclonal antibody used in the detection of adhesins associated with mature endospores of Pasteuria penetrans isolated from Meloidogyne arenaria race 1, and Pasteuria isolates from other phytopathogenic nematodes.
The world-wide destructive capacity of nematodes on cultured plants and consequent loss of crop productivity are well known (Sasser, J. N., Plant Disease 64(1): 36-41, 1987). Chemical nematicides, which have been the control method of choice, are rapidly becoming anathematized because of their real and potential adverse impacts on the environment. The need for the development of alternative chemical or bio-rational control agents of nematodes has been widely stated.
The genus Pasteuria was first described by Metchnikoff in 1888 as a parasite of Daphnidae, with the type species designated as Pasteuria ramosa. Following a lengthy period of relative obscurity and taxonomic confusion, a new species designation, Pasteuria penetrans, was assigned to a bacterial parasite of root-knot nematodes of the genus Meloidogyne (Sayre, R. M., and M. P. Starr, Proceedings of the Helminthology Society, Washington, 52:149-165, 1985). In addition to P. penetrans, other species of Pasteuria have been designated on the basis of spore morphology and host preference. These include Pasteuria nishizawae, a parasite of the cyst-forming nematodes, e.g. Heterodera glycines; Pasteuria thornei, a parasite of lesion nematodes, e.g. Pratylenchus spp., and Pasteuria sp, a parasite of sting nematodes, e.g. Belanolaimus longicaudatus (Chen, Z. X., and D. W. Dickson, Journal of Nematology, 30:313-340, 1998). The physiological aspects of the life cycle of P. penetrans, and its infection of and propagation in root-knot nematodes, is reasonably well understood. The genetic and biochemical aspects are not well understood. The process(es) by which soil-borne endospores become associated with nematodes in the soil involves recognition of the host by carbohydrate-lectin interactions, followed by irreversible attachment. Glycoproteins have been implicated in this process and have been collectively designated as adhesins (Persidis, A., J. G. Lay, T. Manousis, A. H. Bishop, and D. J. Ellar., Journal of Cell Science, 100:616-622, 1991). The nematode may be infected by an attached spore after it has migrated to a feeding site in a root of a host plant and initiated the formation of giant cells characteristic of the root-knot . Proliferation of the bacteria and their development into endospores occurs in the differentiated female nematode, and results in the formation and ultimate release into the soil of as many as 2 million endospores from a single infected nematode.
Furthermore, endospore-forming bacteria assigned to the Pasteuria genus have been recognized as potential agents for the biocontrol of plant-pathogenic nematodes (Sayre, R. M., Plant Disease 4:527-532, 1980; Stirling, O. R., Phytopathology 74:55-60, 1984). Studies at the University of Florida have established an inverse relationship between the levels of endospores of Pasteuria penetrans in the soil and the incidence of infection of peanut by Meloidogyne arenaria (Chen, Z. C., and D. W. Dickson, Journal of Nematology, 30:313-340, 1998). This work has provided the strongest evidence that the levels of endospores of P. penetrans in the soil are responsible for suppressing the nematode infestation of crop plants in the field. It has provided a basis for developing protocols to suppress nematode infestations through increasing the levels of Pasteuria endospores in the soil. Along with several other studies, this work has indicated that levels Pasteuria endospores at certain levels, e.g. greater than 100,000 per g of soil, may be sufficient to provide adequate protection against infection of plants by phytopathogenic nematodes without treatment with chemical nematicides.
Traditionally, the concentration of soil-borne endospores of Pasteuria is determined using a relatively laborious and time-consuming bioassay procedure, wherein cultivated 2nd stage Meloidogyne juveniles (J2) are incubated in a soil sample. The J2 ""s are recovered by centrifugation in sucrose, and the numbers of endospores associated with the nematode cuticle serve as the basis for estimating the number of endospores in the soil sample.
In recent studies at the University of Florida (Gainesville), a monoclonal antibody was developed which was able to bind to intact spores of different isolates of Pasteuria penetrans. The binding of the antibody to the spores prevented the spores from binding to the J2 stage of the root-knot nematode. The monoclonal antibody detected an epitope (determinant) that was shared by several polypeptides derived from the spore. It appears that the epitope is involved in the recognition of endospores and their attachment to the nematode, processes that are necessary for the suppression of the nematode infection of plants. The details of this monoclonal antibody, its method of preparation, and further details of this work are contained in the thesis presented by John H. Charnecki to the Graduate School at the University of Florida, August 1997, for the Master of Science degree and was also referenced in Abstract Q-171, entitled xe2x80x9cDeterminants for the attachment of endospores of Pasteuria penetrans to phytopathogenic nematodesxe2x80x9d, J. H. Charnecki, J. D. Rice, D. W. Dickson, and J. F, Preston on p 449 in the book of abstracts for the 1998 Annual Meetings of the American Society for Microbiology in Atlanta, Ga., May 17 to 21.
Previc et al., U.S. Pat. No. 5,094,954, recognizing the potential use of the bacteria group Pasteuria as biorational control agents against phytopathogenic nematodes, detailed a process for producing endospores of Pasteuria by growing the bacteria on explanted nematode tissue.
There is no way known, other than the referenced method using J2 stage Meloidogyne juveniles, to establish to quality and quantity of the endospores of Pasteuria penetrans produced upon growing the bacteria.outside of the nematode host.
It would therefore be highly useful to have a probe or method that could be used to monitor the levels of Pasteuria spores in the soil: would make possible a measure of the relationship between the levels of spores and the extent to which application of chemical nematicides are needed for the suppression of root-knot and other phytopathogenic nematodes; and would make possible knowledgeable production of Pasteuria spores in quantities adequate for their application as biocontrol agents and alternatives to chemical nematicides.
The first objective of the present invention is to provide a probe for the specific and sensitive detection of endospores of Pasteuria spp.
The second objective of this invention is to provide a method for the accurate and sensitive determination of the levels of endospores of Pasteuria penetrans in soil samples.
The third objective of this invention is to provide a probe for the detection of adhesins associated with the virulence of mature endospores of Pasteuria spp.
A preferred embodiment of the invention is a probe consisting essentially of a monoclonal antibody (MAb) generated against an epitope carried by endospores of Pasteuria penetrans and a method for its use as a probe in detecting the levels of soil-borne Pasteuria endospores comprised of the following steps: mixing a quantity of soil suspected of containing Pasteuria with reagents capable of solublizing the epitope recognized by the MAb; incubating said admixture at a temperature between 37 C and 50 C for a time of 0.5 to 2 hours to allow extraction and solubilization of epitopes; centrifuging to remove debris and provide a soluble preparation of epitopes; quantifying the endospore epitopes with the MAb by ELISA.
Pasteuria penetrans is a Gram-positive, endospore-forming, obligate parasite of phytopathogenic root-Inot nematodes, Meloidogyne spp. Regulations restricting the use of the cropland chemical nematicide, methyl bromide, in the U.S. and elsewhere, has prompted renewed search for alternative methods to control these nematode pests. P. penetrans, as well as other Pasteuria spp., have so far been unamenable to axenic culture and therefore cannot be isolated and quantified by traditional bacteriological methods. A monoclonal antibody (MAb) has been produced against Pasteuria biotype P-20spores, which binds to a putative glycan epitope shared by several polypeptides that are of different molecular masses and that occur in the spore envelope. The epitope detected by this MAb is found in endospores of P. penetrans isolates with a preference for Meloidogyne arenaria race 2, in P. penetrans with preferences for other Meloidogyne spp., and in Pasteuria isolates from other phytopathogenic nematodes. The epitope appears in infected female nematodes at a stage after, but not before, endospore formation can be observed. The disclosure introduces an antigen extraction procedure for soil-borne endospores and a rapid and sensitive ELISA to quantify Pasteuria spores in soil. Linear regression analysis of spore concentrations in soil samples versus absorbance produced good theoretical line fits for all ELISA formats evaluated. A tertiary detection system produced a minimum limit of less than 3000 spores/g of soil tested. Correlation of traditional and immunoassay spore quantitation data produced line fits of 0.99. Immunoblots comparing extracts from isolated spores and soil samples indicate the antigens extracted form the soil share molecular mass identity with those extracted from the isolated spores, providing confirmation that Pasteuria endospore-associated antigens are responsible for the ELISA signal. Cross-reactivity of the MAb extracts of other Gram positive bacteria committed to sporulation was not detected, indicating the epitope detected by the MAb is unique to the endospores of Pasteuria. The presence of the epitope on soil-borne spores and the ability to evaluate levels of P. penetrans as a measure of root-knot nematode infection should mitigate the need for the application of chemical nematicides where the levels of Pasteuria are adequate for the control of phytopathogenic nematode populations.
Further objectives and advantages of this invention will be apparent from the following detailed description of a presently preferred embodiment of a monoclonal antibody for detection of adhesins unique to the endospores of Pasteuria spp., the production of this antibody, and description of applications.
Before explaining the disclosed embodiment of the present invention in detail it is to be understood that the invention is not limited in its application to the details of the particular arrangement shown since the invention is capable of other embodiments. Also, the terminology used herein is for the purpose of description and not of limitation.
A mature Pasteuria bacterium has endospores that are able to infect and suppress the soil levels of root-knot nematodes characterized by adhesins. A monoclonal antibody (MAb) has been produced against Pasteuria biotype P-20 spores, which has the property of binding a putative glycan epitope shared by a variety of molecular weight peptides present as a component of the Pasteuria spore envelope. Endospore and spore is used herein to describe the same entity.
The subject invention is a novel means for detecting the presence of and quantity of mature Pasteuria endospores useful in the biological control of nematodes. According to the subject invention, Pasteuria spores, such as those that infect the root-knot nematode Meloidogyne spp. or other specified host nematodes, are first prepared as monoclonal antibodies in mice that is directed against the P-20 isolate of Pasteuria penetrans by the method
Mouse hybridoma cell line HL 1325 (2A4-1D10) was deposited with the American Type Culture Collection (10801 University Blvd., Manassas, Va. 20110-2209) on Nov. 15, 2001 as accession number PTA-3865.
In order to better understand the production of monoclonal antibodies (MAb) capable of detecting nematode adhesins associated with the presence of the Pasteuria genus, a detailed description of their production is set forth in the prior art referenced Charnecki Thesis (Chapter 2) which is incorporated herein by reference thereto.